Abstract

It has been reported that among drugs with mixed actions on central nervous system monoamine systems, increased serotonergic activity is associated with decreased potency as a reinforcer. The present experiment was designed to examine this relationship for amphetamine analogs that varied in serotonin releasing potency and to evaluate whether serotonergic actions can affect reinforcing efficacy. Compounds PAL 313 and 314 are para- and meta-methylamphetamine, respectively. PAL 303 and 353 are para- and meta-fluoroamphetamine, respectively. All compounds had similar potencies as in vitro releasers of dopamine (DA) and norepinephrine (NE) but differed in potency for 5-hydroxytryptamine (serotonin) (5-HT) release [EC50 (nanomolar) PAL 313 = 53.4; PAL 314 = 218; PAL 303 = 939; PAL 353 = 1937]. When made available to rhesus monkeys (Macaca mulatta)(n = 4) for self-administration under a fixed-ratio 25 schedule, all were positive reinforcers with biphasic dose-response functions (0.003–1.0 mg/kg) and were equipotent. PAL 313 was self-administered at a lower rate than the other compounds, which were indistinguishable. Under a progressive-ratio schedule (n = 5), all drugs were positive reinforcers. Dose-response functions increased to a maximum or were biphasic (0.01–1.0 mg/kg), and drugs were equipotent. At maximum, PAL 313 maintained less responding than other PAL drugs, which maintained similar maxima. Thus, all compounds were positive reinforcers under both schedules, consistent with their potent DA actions. Responding was lower when 5-HT potency was higher and comparable with DA and NE potency. The results suggest that the mechanism for this effect involves a decrease in reinforcing potency and efficacy among monoamine releasing agents when 5-HT releasing potency is increased relative to DA.

Among drugs that enhance monoaminergic neurotransmission in the central nervous system (CNS), especially psycho-stimulants, there is considerable evidence that dopamine (DA) plays an integral part in the reinforcing effects that contribute to their self-administration and abuse (for reviews, see Wise, 1978; Woolverton and Johnson, 1992; Howell and Wilcox, 2002). Observations that are consistent with this hypothesis include the finding that the potency of cocaine-like drugs in self-administration is positively correlated with their affinity in binding at the dopamine transporters (DATs) in vitro (Ritz et al., 1987; Bergman et al., 1989; Wilcox et al., 2000). Simple potency as a reinforcer, however, does not seem to predict potential for abuse particularly well. Rather, abuse has been more strongly associated with efficacy or strength as a reinforcer (Brady and Griffiths, 1976). Cocaine, for example, has a relatively weak affinity at the DAT, but it is one of the most efficacious reinforcers in laboratory animals and is highly abused by humans. It seems that factors other than potency for increasing DA neuro-transmission must contribute to reinforcing efficacy and abuse of compounds that bind the DAT.

Previous research has suggested several pharmacological determinants of reinforcing efficacy. With regard to monoaminergic actions, enhanced serotonergic activity may be negatively related to the self-administration of psychostimulants. Compounds that selectively increase 5-hydroxytryptamine (serotonin) (5-HT) neurotransmission have been found not to maintain self-administration (Tessel and Woods, 1975; Vanover et al., 1992; Howell and Byrd, 1995). Among amphetamine-like drugs that are not as selective for 5-HT activity, Ritz and Kuhar (1989) reported a negative correlation between potency as a reinforcer and binding affinity at the 5-HT transporter (SERT). The depletion of 5-HT by medial forebrain bundle lesion with 5,7-dihydroxy-tryptamine seemed to increase the reinforcing efficacy of cocaine in rats (Loh and Roberts, 1990), whereas manipulations that increase CNS 5-HT function can decrease cocaine self-administration (Smith et al., 1986; Carroll et al., 1990; Howell and Byrd, 1995). Studies using coadministered phentermine and fenfluramine as a prototypical DA/5-HT releasing agents demonstrated that compounds that increase both extracellular DA and 5-HT in rat nucleus accumbens are not self-administered by rodents (Glatz et al., 2002), do not induce conditioned place preference in rats (Rea et al., 1998), and have low potential for abuse in human subjects (Brauer et al., 1996). Reinforcing efficacy among a series of cocaine analogs was shown to be negatively related to the SERT potency relative to DAT (Roberts et al., 1999). In that study, cocaine analogs with the highest relative SERT affinity did not function as reinforcers.

Together, these data suggest that among drugs that increase monoaminergic neurotransmission in the CNS, 5-HT activity may reduce reinforcing efficacy and thereby reduce self-administration. In a recent study with rhesus monkeys (Macaca mulatta), Lile et al. (2003) reported robust self-administration of a cocaine analog, HD-60, with at least 80-fold selectivity for the SERT relative to the DAT. This same compound was not a positive reinforcer in rats in the study of Roberts et al. (1999) (called WF-60). Thus, it is possible that there are important species differences in the influence of 5-HT activity on self-administration. In addition, although Ritz and Kuhar (1989) proposed a negative relationship between 5-HT release and reinforcing potency, a prospective study testing this hypothesis, or the relationship of 5-HT release to reinforcing efficacy, has not been reported. The present experiments were therefore designed to evaluate the relationship between 5-HT releasing potency, relative to other monoamines, and reinforcing potency and efficacy in rhesus monkeys. A series of amphetamine analogs with similar in vitro potencies in releasing DA and NE, but varying potencies in releasing 5-HT, were selected for testing.

Materials and Methods

All animal use procedures were approved by the University of Mississippi Medical Center's Animal Care and Use Committee and were in accordance with National Institutes of Health guidelines.

Self-Administration Studies

Animals and Apparatus. The subjects were seven male rhesus monkeys [88-14, AP01, AP78, and M341 for the fixed-ratio (FR) study; AP01, M341, M1389, L500, and RJu2 for the progressive-ratio (PR) study], weighing between 9.6 and 11.8 kg at the beginning of the study. All the monkeys except 88-14 had histories of self-administration of cocaine and/or other stimulants. Monkey 88-14 had previously been trained in a drug discrimination paradigm to discriminate pentobarbital from saline and was naive to drug self-administration. Monkey Rju2 became ill and had to be removed from the study before all drugs had been tested. All monkeys were provided with sufficient food to maintain stable body weight (120–180 g/day; Teklad 25% Monkey Diet; Harlan/Teklad, Madison, WI) and had unlimited access to water. Fresh fruit and a vitamin supplement were provided daily and three times a week, respectively. Lighting was cycled to maintain 16 h of light and 8 h of dark, with lights on at 6:00 AM.

The monkeys were individually housed in the experimental cubicles (1.0 m3; Plaslabs, Lansing, MI). Each monkey was fitted with a stainless steel harness attached by a tether to the rear wall of the cubicle. The front door of the cubicle was made of transparent plastic and the remaining walls were opaque. Two response levers (PRL-001; BRS/LVE, Beltsville, MD) were mounted on the inside of the door. Four jeweled stimulus lights, two red and two white, were mounted above each lever. Drug injections were delivered by a peristaltic infusion pump (Cole-Parmer Instrument Co., Chicago, IL). A Macintosh computer with custom interface and software controlled all events in an experimental session.

Procedure. Monkeys were implanted with a silastic catheter (0.26 cm o.d. × 0.076 cm i.d.; Cole-Parmer Instrument Co.) into the jugular (internal or external) or femoral vein under isoflurane anesthesia. Brachial veins were implanted with a microrenethane catheter (0.08 in. o.d. × 0.04 in. i.d.; Braintree Scientific, Braintree, MA) heated and drawn to approximately half size. The proximal end of the catheter was inserted into the vein and terminated in the vena cava near the right atrium. The distal end was threaded subcutaneously to exit the back of the monkey, threaded through the spring arm, out the rear of the cubicle, and connected to the peristaltic pump. In the event of catheter failure, surgery was repeated using another vein, after the veterinarian confirmed the health of the monkey.

Experimental sessions began at noon each day and were conducted 7 days per week. Thirty minutes before each session started, catheters were filled with drugs for the sessions without infusing the drugs into monkeys. At the start of a session, the white lights were illuminated above both levers and pressing the right lever resulted in the delivery of a drug injection for 10 s. During the injection, the white lights were extinguished and the red lights were illuminated. Pressing the left lever was counted but had no other programmed consequence. After the session, catheters were filled with 0.9% saline containing heparin (40 units/ml).

In baseline sessions, cocaine or saline was available for an injection. The baseline dose of cocaine or saline was initially available under a double-alternation schedule, i.e., two consecutive daily cocaine sessions were followed by two consecutive daily saline sessions. When responding was stable (see below) for at least two consecutive double-alternation sequences of cocaine and saline (i.e., eight sessions), test sessions were inserted to the daily sequence between two saline or two cocaine sessions. To prevent monkeys from learning this session sequence, a randomly determined saline or cocaine baseline session was inserted after every other test session. Thus, the daily sequence of sessions was C, S, T, S, C, T, R, C, S, T, S, C, T, R, where C, S, R, and T represent a cocaine baseline, a saline, a randomly determined cocaine/saline, and a test session, respectively.

Drugs were tested in a different order across monkeys. All doses of one compound were tested before moving on to the next compound. For the first monkey tested with a given drug, doses were available in an ascending order. For the other monkeys, doses were tested in a random order. After a test session, a monkey was returned to baseline conditions until responding for cocaine and saline again met stability criteria or until a new stable baseline was established. All doses were tested at least twice in each monkey, once with a saline session the day before and once with a cocaine session the day before.

Fixed-ratio schedule. Drugs were made available under an FR schedule that has been described previously (Wee and Woolverton, 2004). The response requirement was 25 lever presses per injection, and each session lasted for 2 h. The baseline dose of cocaine was the dose that maintained the maximum responding in a dose-response function in all monkeys, i.e., 0.03 mg/kg/injection. Responding was considered stable when the injections/session for cocaine varied no more than ±15% of the previous session and saline injections were less than 15 injections/session, and there were no trends in the data. During test sessions, one of various doses of the amphetamine analogs (0.003–1.0 mg/kg) was made available for self-administration under conditions identical to baseline conditions. When the data in the two test sessions of a given dose were inconsistent (i.e., a dose was a reinforcer in one test and not in the other or a reinforcer in both tests but intake differed by more than 30 injections), the dose was made available for at least four consecutive sessions and until responding was stable.

Progressive-ratio schedule. Drugs also were made available to a second group of monkeys in which responding was maintained under a PR schedule of reinforcement comparable with that described by Wilcox et al. (2000). The PR schedule consisted of 20 trials, with one injection available per trial. The response requirement started at 100 responses per injection and doubled after every fourth trial. A subject had 30 min to complete a trial [limited hold (LH) 30 min]. A trial ended with a 10-s drug injection or the expiration of the LH. There was a 30 min timeout (TO) after each trial. If the response requirement was not completed for two consecutive trials (i.e., the LH expired) or the animal self-administered all 20 injections, the session ended.

The baseline dose of cocaine was the lowest dose that maintained the maximum injections in individual monkeys, i.e., 0.1 or 0.3 mg/kg/injection. Responding was considered stable in baseline sessions when injections/sessions varied by no more than two for both cocaine and saline for at least two consecutive double-alternation sequences. During test sessions, one of various doses of amphetamine and the amphetamine analogs (0.003–1.0 mg/kg) was available for monkeys under conditions identical to baseline sessions. When the two test sessions of a dose showed high variability (two determinations ≥ mean ± three injections) at the dose with the maximum mean injections, the dose was redetermined twice, once after saline session and once after cocaine baseline session. For drugs that exhibited biphasic dose-response functions, the TO after injection was increased to 60 min, and the two highest doses were tested again. This adjustment was made out of concern that, under the 30-min TO, drug accumulation over the session may have acted to suppress responding and thereby decreased maximum responding.

Data Analysis. The mean number of injections per session was calculated individually from the two test sessions as a function of dose. When a dose was tested in consecutive sessions under the FR schedule, the last two test sessions were used in data analysis. When a dose was retested under the PR schedule, the two retest sessions were used in data analysis. The range of injections served as a measure of variability in individual subjects. A dose of a drug was considered to function as a reinforcer if the mean number of injections was above levels seen with saline, and the ranges did not overlap.

For both schedules, the group mean dose-response functions for each drug were collapsed across the monkeys as to the dose of the maximum injections, regardless of the absolute values of the doses. This was done because each drug maintained qualitatively comparable dose-response functions across monkeys but with the maximum responding at a different dose, in particular under the FR schedule. Statistical analysis was done with these group means normalized as to dose. That is, the mean maximum number of injections was calculated for a drug by averaging the individual maximum mean injections for the drug, regardless of dose, across monkeys (Woolverton and Wang, 2004). Repeated measures one-way analysis of variance (ANOVA) with the Student-Newman-Keuls as a post hoc test was then used to assess statistically significant differences among drugs in the maximum number of injections (GraphPad Prism 3.0; GraphPad Software Inc., San Diego, CA).

Under the FR schedule, the potency of the compounds in self-administration was compared based on the doses that maintained the maximum injections in monkeys in which a drug served as a reinforcer. The doses of the maximum injections in individual monkeys were averaged for a given drug and compared using a repeated measures one-way ANOVA.

For the data under a PR schedule, the ED50 value was calculated for each animal in which a drug served as a reinforcer using the ascending limb of a dose-response function and nonlinear regression analysis (GraphPad Prism 3.0). Mean ED50 values were calculated for each drug by averaging the log values of ED50 values in all monkeys in which the drug functioned as a reinforcer and taking the antilog of that value. Repeated measures one-way ANOVA, using multiple imputation to fill empty ED50 values in two monkeys for statistical purpose, was used to compare ED50 values (GraphPad Prism 3.0). The Student-Newman-Keuls was used as a post hoc test.

In Vitro Monoamine Release

In vitro release assays were conducted as described by Rothman et al. (2003) using [3H]MPP+ as the radioligand for both the DA and NE release assays. Rat caudate (for DA release) or whole brain minus cerebellum and caudate (for NE and 5-HT release) was homogenized in ice-cold 10% sucrose containing 1 μM reserpine. Nomifensine (100 nM) and GBR12935 (100 nM) were added to the sucrose solution for [3H]5-HT release experiments to block any potential [3H]5-HT reuptake into NE and DA nerve terminals. For the DA release assay, 100 nM desipramine and 100 nM citalopram were added to block [3H]MPP+ uptake into NE and 5-HT nerves. For the NE release assay, 50 nM GBR12935 and 100 nM citalopram were added to block [3H]MPP+ uptake into DA and 5-HT nerves. After 12 strokes with a Potter-Elvehjem homogenizer, homogenates were centrifuged at 1000g for 10 min at 0 to 4°C, and the supernatants were retained on ice (synaptosomal preparation).

Data Analysis and Statistics. As described previously (Rothman et al., 1993), EC50 values for transporter assays were determined using the nonlinear least-squares curve fitting program MLAB-PC (Civilized Software, Bethesda, MD). A correlation between in vitro measures and potency as a reinforcer under both FR and PR schedules or between in vitro measures and the maximum injections under both FR and PR schedules was evaluated using Pearson correlation coefficient (GraphPad Prism 3.0 or Primer of Biostatistics). When the data violated the assumptions for parametric analysis (i.e., equal variance), Spearman correlation coefficient was used.

Drugs

Cocaine hydrochloride and amphetamine sulfate were provided by the National Institute on Drug Abuse (Rockville, MD). Amphetamine analogs PAL 314 (m-methylamphetamine), PAL 313 (p-methylamphetamine), PAL 353 (m-fluoroamphetamine), and PAL 303 (p-fluoroamphetamine) (Fig. 1) were synthesized using methodology described by Monte et al. (1997). The appropriate aldehydes were converted to their nitro-olefins using ammonium acetate and nitroethane. These crude nitro-olefins were subsequently reduced with lithium aluminum hydride to obtain the desired compounds. Each compound was purified from the crude material as either a hydrochloride or fumarate salt. Purity was confirmed by combustion analysis and 1H NMR. For the self-administration study, drugs were dissolved in 0.9% saline. Doses were expressed as the salt forms of the drugs.

Chemical structures of amphetamine and the substituted amphetamines tested.

Results

Self-Administration under a Fixed-Ratio Schedule. In test sessions, the baseline dose of cocaine (0.03 mg/kg/injection) maintained a mean of 54 injections/session (S.E.M. 10.3), whereas saline maintained a mean of four injections/session (S.E.M. 1.5; Fig. 2). All PAL compounds maintained responding above saline levels at least at one dose in all monkeys, and dose-response functions were biphasic. Monkey 88-14 was reliably more sensitive to all drugs and consistently responded at higher rates than the other monkeys. Based upon the dose that maintained maximum responding, the PAL compounds did not differ significantly in potency [Table 1; F(3,9) = 2.72; p > 0.1]. The mean maximum responding maintained by PAL 313 was significantly lower than that for PAL 314, PAL 303, and PAL 353 [Fig. 2; F(3,12) = 6.13; p < 0.05].

Self-administration of PAL compounds under an FR25 schedule of reinforcement. Drugs were available for self-administration for 2 h/day. Each data point represents the mean injections/session of each dose for four rhesus monkeys, and vertical error bars represent the S.E.M. values. The point above Sal or Coc represents self-administration of saline or the baseline dose of cocaine in test sessions, respectively. Data were normalized as to dose to adjust for individual differences in sensitivity. Max, dose that maintained maximum injections in each animal; Max –1, half-log dose lower than Max; Max+1, half-log dose higher than Max. *, p < 0.05 compared with PAL 314, PAL 303, or PAL 353. Inset, dose-response functions of PAL compounds. The drugs were tested in a different dose range in one monkey because of different sensitivity to the drugs. Thus, data points are the mean of three or four monkeys. When the data point was from less than three monkeys, the number of subjects is indicated in parentheses.

Potencies and maximum responding of PAL compounds in self-administration

Data are mean ± S.E.M., and numbers in parentheses are numbers of animals tested. For the FR schedule, the mean dose that maintained responding at the peak of the biphasic curve was calculated for potency. For the PR schedule, an ED50 dose of a dose-response function was obtained in individual monkey and averaged across the monkeys for the mean. Potency is expressed as micromoles per kilogram per injection. For the PR potency, log mean ED50 (milligrams per kilogram per injection) ± S.E.M. was added in parentheses to indicate variation. The maximum injection indicates the maximum injections per session.

Self-Administration under a Progressive-Ratio Schedule. The baseline dose of cocaine (0.1 or 0.3 mg/kg/injection) maintained a mean of 15.2 injections/session (S.E.M. 1.1), whereas saline maintained a mean of 1.6 injections/session (S.E.M. 0.2; Fig. 3). d-Amphetamine and PAL 303, 314, and 353 functioned as positive reinforcers in all monkeys. PAL 313 functioned as a positive reinforcer in four of five monkeys (L500, M1389, AP78, and RJu2). Monkey M341 initially showed high variability at two of four doses of PAL 313. When retested, this monkey did not self-administer PAL 313 at any dose. Dose-response functions increased with dose over low-to-moderate doses and were asymptotic or decreased again at higher doses. PAL 313, PAL 314, PAL 303, and PAL 353 did not differ significantly in potency, and d-amphetamine was approximately 6- to 12-fold more potent [Table 1; F(4,16) = 17.7; p < 0.0001]. At maximum, d-amphetamine maintained more injections/session than the PAL compounds, whereas PAL 313 maintained fewer injections/session than the other compounds [Fig. 3; F(3,12) = 585; p < 0.001]. The maximum number of injections of PAL 314, PAL 303, and PAL 353 were indistinguishable. When drugs with biphasic dose-response functions, amphetamine, PAL 303, and PAL 353 were reexamined with a TO of 60 min, the shapes of the dose-response functions were unchanged; i.e., they were asymptotic or biphasic. Furthermore, the maximum number of injections did not exceed those seen with the shorter TO for any drug.

Self-administration of d-amphetamine and PAL compounds under a progressive-ratio schedule of reinforcement. Each data point represents the mean injections/session for four (d-amphetamine) or five monkeys (all PAL compounds), and vertical error bars represent the S.E.M. values. The point above Sal or Coc represents self-administration of saline in test sessions or cocaine during baseline sessions, respectively. Data were normalized as to dose to adjust for individual differences in sensitivity. Max, dose of the maximum injections in each animal. Max-1, half-log dose lower than Max; Max-2, full-log dose lower than Max. ***, p < 0.001 compared with each of the other compounds. Inset, dose-response function of amphetamine and PAL compounds. The drugs were tested in different dose ranges in monkeys because of different sensitivities to the drugs. Thus, data points are the mean of four or five monkeys. When the data point was pooled from less than four monkeys, the number of subjects is indicated in parentheses.

In Vitro Monoamine Release Assay.d-Amphetamine and the PAL compounds released DA, NE, and 5-HT in a concentration-dependent manner, and concentration-effect functions were parallel (data not shown). All the compounds showed comparable potencies releasing DA with the EC50 values between 8.0 and 51.5 nM (Table 2). Similarly, the EC50 values for the PAL compounds did not differ for NE release with the EC50 values between 7.2 and 28.0 nM (Table 2). As for 5-HT release, the order of the potencies was PAL 313 > PAL 314 > PAL 303 > PAL 353 > d-amphetamine. Thus, the DA/NE potency ratio ranged between 1.1 and 2.0, whereas the DA/5-HT potency ratio ranged between 0.004 and 0.83.

Relationship between Self-Administration and in Vitro Effects. For the FR schedule, there was no significant correlation between potency as a reinforcer (Table 1) and in vitro potency (Table 2) releasing DA (r = 0.55, df = 3, p = 0.34) or 5-HT (r =–0.84, df = 3, p = 0.08). However, potency as a reinforcer was positively correlated with the ratio of the in vitro potencies releasing DA/5-HT (r = 0.98, df = 3, p = 0.004). There was no correlation with the ratio of the in vitro potencies releasing DA/NE (r = 0.78, df = 3, p = 0.12). Maximum responding under the FR schedule was not correlated with any measure of in vitro potency. For the PR schedule, there was also a positive correlation between the ratio of the in vitro potencies releasing DA/5-HT and reinforcing potency (r = 0.97, df = 4, p = 0.02). However, potency as a reinforcer was not correlated with any other measure of in vitro potency. Efficacy as a reinforcer under the PR schedule was not correlated with any measure of in vitro potency.

Discussion

As predicted based upon their nanomolar potencies as releasers of DA, all of the tested compounds functioned as positive reinforcers in all monkeys responding under an FR schedule of reinforcement. Dose-response functions under the FR schedule were biphasic, as is typically seen under these conditions (Young and Herling, 1986; Bergman et al., 1989). Although not tested in the present study, d-amphetamine has been found to function as a reinforcer under conditions similar to those used here (Woolverton et al., 2001). Additionally, all but one of the compounds, PAL 313, served as a positive reinforcer in all monkeys responding under a PR schedule. PAL 313 was a reinforcer in four of the five monkeys tested. The compounds did not differ in potency in either self-administration assay and had comparable potencies as DA releasers in vitro, results that are at least consistent with the conclusion that DA release is involved in their reinforcing effects. Although a similar argument could be made for NE, NE agonists have not previously been reported to function as positive reinforcers (Woolverton, 1987; Wee and Woolverton, 2004). The reinforcing effects of these compounds in the present experiment are consistent with full or partial amphetamine-like discriminative stimulus effects that have been reported previously for PAL 303, PAL 313, and m-methylamphetamine (Higgs and Glennon, 1990; Marona-Lewicka et al., 1995). Together, these results suggest amphetamine type of abuse potential of the compounds.

The lack of a significant negative correlation between potency as a reinforcer under the FR schedule and potency for releasing 5-HT in vitro is inconsistent with the hypothesis proposed by Ritz and Kuhar (1989). It is worth noting, nevertheless, that the correlation for this relationship approached statistical significance (p = 0.08) and may have achieved statistical significance with additional degrees of freedom. Additionally, the most potent 5-HT releaser, PAL 313, was clearly less potent than the least potent 5-HT releaser, d-amphetamine. As an alternative view, the correlation between the ratio of DA/5-HT releasing potency in vitro and potency as a reinforcer was significant, suggesting that the mix of dopaminergic and serotonergic actions is a determinant of potency as a reinforcer. Results were similar for the PR schedule in that potency as a 5-HT releaser was not correlated with potency as a reinforcer among the PAL compounds, although again the least potent 5-HT releaser d-amphetamine was the most potent reinforcer. As for the FR schedule, the ratio of DA/5-HT in vitro potencies predicted potency as a reinforcer under the PR.

The compounds differed in the maximum rate of self-administration under the FR schedule, to the extent that rate of responding was lower for PAL 313 than for the other compounds. PAL 313 was the most potent in vitro 5-HT releaser and had the highest in vitro DA/5-HT potency ratio. Although this result raises the possibility of a relationship between DA/5-HT activity and reinforcing efficacy, this conclusion cannot be drawn unambiguously. Rate of self-administration under a simple FR schedule is determined not only by the reinforcing effect of a drug but also by other effects of the drug that can affect rate. These have been termed “nonspecific” effects and may include effects on motor, sensory, or integrative function; satiety; or punishing effects. Any or all of these might be influenced by an increase in 5-HT activity. The PR schedule, by increasing response requirement for successive injections, measures maximum behavioral output maintained by an injection. From this information an estimate of a drug's efficacy as a reinforcer, or its maximum reinforcing effect, can be inferred. The present PR, by arranging a TO after each injection, is designed to minimize the influence of nonspecific effects on responding. The TO allows these effects to dissipate, at least partially, between injections. Results from the PR schedule are consistent with a conclusion that the reinforcing efficacy of each of the PAL compounds was lower than that of d-amphetamine. Moreover, the reinforcing efficacy of PAL 313 was lower than that of the other compounds. This efficacy relationship was directly related to 5-HT potency, with d-amphetamine the least potent and PAL 313 the most potent 5-HT releaser. The implication is that increased potency as a 5-HT releaser is associated with diminished reinforcing efficacy.

Overall, results from both schedules of reinforcement lend support to the hypothesis that potency as a 5-HT releaser contributes negatively to potency as a reinforcer, but with the refinement that the DA/5-HT potency ratio may be a better predictor than 5-HT potency alone. The observation that the compound with the highest 5-HT releasing potency (PAL 313) was the least efficacious reinforcer, whereas the compound with the lowest 5-HT releasing potency (d-amphetamine) was the most efficacious reinforcer, supports the conclusion of Roberts et al. (1999) for DA uptake blockers. The observation that the compound (PAL 313) with the highest 5-HT potency had the lowest potency and efficacy as a reinforcer, whereas the other PAL compounds did not apparently differ on these measures, raises the possibility that there may be a threshold of 5-HT activity for these effects to be evident. Additional research would be required to determine whether this threshold is for absolute 5-HT potency or 5-HT potency relative to DA potency.

There are at least two possible accounts of the reduced reinforcing efficacy of compounds with increased 5-HT/DA potency. It is possible that increased 5-HT release opposes the activation of DA systems that are involved in the reinforcing effect. Such an effect would not be apparent in the in vitro results, but it could be measured in vivo. Indeed, in an experiment using in vivo microdialysis in rats (Clark et al., 2004), PAL 313 showed diminished efficacy for increasing extracellular DA relative to the other compounds, even with comparable DA releasing potency in vitro. In research with monoamine uptake blockers, Howell and colleagues have published data consistent with an opposing effect of 5-HT activity on DA activity. More specifically, cocaine-induced increases in extracellular DA concentrations, measured in vivo, were diminished after pretreatment with the selective 5-HT reuptake blocker alaproclate in monkeys (Czoty et al., 2002). Alaproclate also decreased the metabolic effects of cocaine in monkeys, as measured by positron emission tomography (Howell et al., 2002). The present results are consistent with the conclusion that 5-HT actions oppose DA actions involved in reinforcing effects.

The possibility also should be considered that 5-HT activity could act to decrease self-administration of a DA agonist via a punishment mechanism. It has been demonstrated that i.v. drug injections can punish ongoing behavior maintained by food delivery (Katz and Goldberg, 1986; Woolverton, 2003). Delivery of response contingent electric shock also can decrease self-administration of cocaine (Bergman and Johanson, 1981). In preliminary experiments (W. L. Woolverton, unpublished data), the addition of histamine to an injection of cocaine can act to punish cocaine self-administration. Considering the clinical reports of unpleasant side effects of drugs that increase 5-HT neurotransmission (Sternbach, 1991; Birmes et al., 2003; Finfgeld, 2004), it is conceivable that the addition of 5-HT actions could suppress self-administration of a DA agonist.

It is interesting to note that PAL 313 was approximately equipotent in releasing all three monoamines. In this effect, it parallels cocaine, which is equipotent as a blocker of uptake of DA, NE, and 5-HT in vitro. Although cocaine dose-response functions were not determined in the present experiments, cocaine has been repeatedly tested under these conditions (Wilcox et al., 2000; Woolverton and Wang, 2004) and is clearly a more efficacious reinforcer than PAL 313. The reason(s) for this difference is unclear, although it does indicate the relationship between monoamine activity and reinforcing efficacy is not simple. This conclusion is underscored by the reports, noted previously, of self-administration of HD-60 by monkeys (Lile et al., 2003) but not by rats (Roberts et al., 1999). HD-60 is a high-potency 5-HT uptake blocker relative to its potency as a DA uptake blocker. It has been reported that monoamine releasers can produce a robust increase in the extracellular monoamines in vitro, whereas reuptake transporter inhibitors of similar potencies were less effective (Rothman and Baumann, 2002). Monoamine transporter inhibitors depend on the basal firing rates of neurons to increase extracellular monoamines, and at the same time, seem to stimulate the negative feedback inhibition, actions that may contribute to a relatively smaller increase in the neurotransmitters. This difference may contribute to the greater influence of serotonergic activity on the self-administration of PAL 313 than on the self-administration of cocaine.

Acknowledgments

We acknowledge the expert advice of Dr. Warren May on the statistical analysis and the technical assistance of Hamilton McGee, Stephanie Dyson, and Joy Flenner.

Footnotes

This study was supported by National Institute on Drug Abuse Grants DA-10352 (to W.L.W.) and DA-12970 (to B.E.B.). W.L.W. is the recipient of National Institute on Drug Abuse Grant K05-DA15343. This work was previously presented at the 2004 meeting of the College on Problems of Drug Dependence, 12–17 June 2004, San Juan, Puerto Rico.

Wilcox KM, Rowlett JK, Paul IA, Ordway GA, and Woolverton WL (2000) On the relationship between the dopamine transporter and the reinforcing effects of local anesthetics in rhesus monkeys: practical and theoretical concerns. Psychopharmacology153:139–147.